Genetic Characteristics of Extended-Spectrum Beta-Lactamase-Producing Salmonella Isolated from Retail Meats in South Korea

Earlier studies have validated the isolation of extended-spectrum beta-lactamase-producing Salmonella (ESBL-Sal) strains from food. While poultry is recognized as a reservoir for Salmonella contamination, pertinent data regarding ESBL-Sal remains limited. Consequently, the Ministry of Food and Drug Safety has isolated Salmonella spp. from retail meat and evaluated their antibiotic susceptibility and genetic characteristics via whole-genome sequencing. To further elucidate these aspects, this study investigates the prevalence, antibiotic resistance profiles, genomic characteristics, and homology of ESBL-Sal spp. obtained from livestock-derived products in South Korean retail outlets. A total of 653 Salmonella spp. were isolated from 1,876 meat samples, including 509 beef, 503 pork, 555 chicken, and 309 duck samples. The prevalence rates of Salmonella were 0.0%, 1.4%, 17.5%, and 28.2% in the beef, pork, chicken, and duck samples, respectively. ESBL-Sal was exclusively identified in poultry meat, with a prevalence of 1.4% in the chicken samples (8/555) and 0.3% in the duck samples (1/309). All ESBL-Sal strains carried the blaCTX-M-1 gene and exhibited resistance to ampicillin, ceftiofur, ceftazidime, nalidixic acid, and tetracycline. Eight ESBL-Sal isolates were identified as S. Enteritidis with sequence type (ST) 11. The major plasmid replicons of the Enteritidis-ST11 strains were IncFIB(S) and IncFII(S), carrying antimicrobial resistance genes (β-lactam, tetracycline, and aminoglycoside) and 166 virulence factor genes. The results of this study provide valuable insights for the surveillance and monitoring of ESBL-Sal in South Korean food chain.


Introduction
Salmonella is a predominant etiological agent of diarrhea, constituting a pervasive global health concern with an annual incidence of 1.9 billion cases worldwide [1].Moreover, its substantial contribution to diarrheal diseases surpasses that of other enteric pathogens [2].The main sources of Salmonella infection include contaminated water, eggs, and meats [3].Contaminated meat is also an important reservoir of antibiotic-resistant genes (ARGs) [4].The dissemination of antibiotic-resistant bacteria (ARB) through the food chain poses a substantial risk as it can introduce these pathogens into the human gastrointestinal tract [5].Therefore, meat contaminated with antibiotic-resistant Salmonella has emerged as a significant threat to human health worldwide.
Various antibiotics have been extensively employed for disease prevention and treatment in the livestock industry globally [6].Notably, fluoroquinolones and cephalosporins have been prominently utilized for treating Salmonella infections [7].Simultaneously, there has been a discernible increase in the application of beta-lactam antibiotics for treating Salmonella infections [8].This heightened usage can exert a selective pressure conducive to the proliferation of extended-spectrum beta-lactamase (ESBL) bacteria [9].Certain ESBL genes, located on plasmids or prophages, facilitate horizontal gene transfer to non-ESBL bacteria [4,5].Consequently, food contaminated with ESBL-producing Salmonella (ESBL-Sal) may serve as a reservoir for the spread of antimicrobial resistance (AMR) within the local community.
Previous studies have demonstrated that ESBL-Sal strains can be isolated from food samples [10][11][12][13].Although poultry is widely acknowledged as a main source for Salmonella contamination, the available data on ESBL-Sal in this context remains limited [12,14].Therefore, to address this gap, the Ministry of Food and Drug Safety (MFDS) has isolated Salmonella spp.from retail meat samples, assessed their susceptibility to antibiotics, particularly betalactams, and comprehensively characterized their genetics through whole-genome sequencing (WGS).In pursuit

ESBL Production Test
For cases exhibiting ceftiofur resistance and ceftazidime MIC ≥ 2 μg/mL, the ESBL phenotype was analyzed via broth microdilution using the ESB1F panel (TREK Diagnostic Systems) following the manufacturer's instructions.The ESBL phenotype strain was characterized by an eight-fold reduction in cefotaxime and ceftazidime MICs when tested in combination with clavulanate, compared to the MICs observed in the absence of clavulanate.

Whole Genome Sequencing and Sequence Analysis
Confirmed ESBL phenotype strains (n = 9) were subjected to WGS at Senigen Inc. (SRepublic of Korea).WGS was performed to determine serotypes, ARGs, plasmid replicons, multilocus sequence typing (MLST), core genome MLST (cgMLST), and virulence genes.Briefly, bacterial genomic DNA was extracted using the UltraClean Microbial DNA Isolation Kit (MoBio Laboratories Inc., USA) following the manufacturer's instructions.Sequencing was performed on an Illumina MiSeq desktop sequencer (Illumina Inc., USA).WGS was performed using 300 bp paired-end sequencing.Raw reads were assembled using the SPAdes genome assembler version 3.13.0.Contigs less than 200 bp length and 5× sequencing depth were removed.Assembled contigs maintained an average sequencing depth of 130×.

Phylogenetic Analysis
Homology among ESBL phenotype strains was compared using MLST and cgMLST.The MLST database (http://pubmlst.org/database/)referenced seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, and thrA), and MLST 2.0 assigned sequence types to strains.For cgMLST analysis, raw sequence data files were uploaded to cgMLSTfinder 1.2 at the Center for Genomic Epidemiology [17], and allelic profiles were predicted.A minimum spanning tree based on the allelic profile of cgMLST was constructed using GrapeTree version 1.5.0.

In Silico Characterization of WGS
ARGs were predicted using ResFinder 4.1, with 90% as the minimum for identity and 60% as the cut-off query length.Plasmid typing was predicted using Plasmidfinder 2.1, with 95% as the minimum for identity and 60% as the cut-off query length.Salmonella pathogenicity islands were predicted using SPIFinder 2.0, with 95% as the minimum for identity and 60% as the minimum for coverage.Salmonella serotypes were predicted using SeqSero 1.2.Virulence factors were identified using the Virulence Factor Database [18], with 90% as the minimum for identity and 50% as the minimum for coverage.

Statistical Analysis
Chi-square tests on the Epitools website were employed to assess the significance of proportional differences within the 95% confidence interval scale [19].Briefly, we compared Salmonella isolates from chicken and duck samples, specifically examining antibiotic-resistant and antibiotic-sensitive strains.

Prevalence of ESBL-Producing Salmonella in Meat Samples
A total of 191 Salmonella strains were isolated from 1,864 meat samples.The prevalence rates of Salmonella were 28.2%, 17.5%, and 1.4% in the duck, chicken, and pork samples, respectively.No Salmonella strain was isolated from beef.Furthermore, the prevalence of ESBL-Sal was 1.4% in the chicken samples and 0.3% in the duck samples (Table 2).Only nine strains were identified as ESBL-producing Salmonella.Thus, poultry meat exhibited a higher prevalence of Salmonella and ESBL-Sal.

Antimicrobial Resistance Patterns of Salmonella
A chi-square test (p < 0.05) revealed significant differences in AMR between chicken and duck isolates for ten antibiotics, namely, amoxicillin/clavulanic acid, ampicillin, cefepime, ceftiofur, ceftazidime, nalidixic acid, streptomycin, sulfisoxazole, tetracycline, and trimethoprim/sulfamethoxazole (Table 3).Among poultry samples,  the most prevalent resistance to non-beta lactam antibiotics was observed with nalidixic acid and tetracycline.Salmonella strains isolated from pork were resistant to only four antibiotics: ampicillin, streptomycin, sulfisoxazole, and tetracycline.

Phylogenetic Analysis
The homology among the nine ESBL-Sal isolates was assessed using cgMLSTFinder (version 1.2) and GrapeTree (version 1.5.0)(Fig. 1).In the minimum spanning tree, these nine ESBL-Sal isolates were divided into one cluster and one singleton.The singleton, identified as the strain 2019_258 chicken isolate, was assigned cgST96964 and ST16.The cluster comprised eight strains (2018_11, 2018_64, 2018_452, 2018_800, 2018_916, 2018_963, 2019_259, and 2019_265), all predicted to be cgST58360, ST11, and S. Enteritidis.These cluster strains were isolated from seven chickens and one duck.Notably, an average of 2,499 allelic differences were observed between the cluster and singleton.

Discussion
In this study, we isolated 97 and 87 Salmonella strains from 555 chicken and 309 duck samples, respectively.The prevalence of Salmonella was 17.5% in the chicken samples and 28.2% in the duck samples.The chicken isolates exhibited resistance rates of 22.7% and 21.6% to ceftiofur and ceftazidime, respectively.However, the duck isolates of Salmonella exhibited resistance rates of 2.3% and 2.3% to ceftiofur and ceftazidime, respectively.The prevalence of ESBL-Sal was 1.4% in the chicken samples and 0.3% in the duck samples, respectively.Samples were collected between 2018 and 2019, with a roughly equal distribution of samples collected repeatedly.However, substantial disparities were observed in the prevalence rate of Salmonella, ESBL-Sal, and 3rd-generation cephalosporin AMR between the chicken and duck meat samples.The introduction of antibiotics decreased microbial diversity; high antibiotic selective pressures increased the abundance of ARGs but coincided with a reduction in the diversity of ARGs [20].These findings suggest a higher likelihood of Salmonella contamination with a high AMR level in chicken meat than in duck meat, indicating a potentially greater use of antibiotics in chicken production.
In this study, S. Enteritidis was considered a representative serotype from poultry, which may cause ESBL-Sal infections in humans.The ESBL-Sal strain isolated from poultry meat in most parts of the country exhibited a specific sequence type and serotype of Enteritidis-ST11.This predominant group displayed one cluster in the cgMLST analysis.Moreover, these strains exhibited resistance to ampicillin, ceftiofur, ceftazidime, nalidixic acid, and tetracycline.Genomic characterization revealed the presence of beta-lactam, tetracycline, and aminoglycoside resistance genes in two plasmid replicons, namely, IncFIB(S) and IncFII(S).WGS analysis revealed 11 identical pathogenicity islands and 156 identical virulence factor genes.
This study has certain limitations.The study design was limited in the context of the sampling of ESBL-Sal isolates.Utilizing a Salmonella selective culture medium, we isolated a maximum of one Salmonella spp.per meat sample.Consequently, this study may differ from studies that used an ESBL-selective culture medium to isolate ESBL bacteria.Moreover, most Salmonella serotypes could not be determined.
Despite these limitations, this study provides valuable insights into the WGS characteristics of ESBL-Sal in South Korean retail meat.The use of MLST and cgMLST allowed for homology determination, which could enable traceback investigations in ESBL-Sal poisoning.Additionally, we identified various major genes, including those related to AMR, plasmid replicon, identical pathogenicity island, and virulence factor genes.Ultimately, this research offers valuable information that can aid in preventing the continued spread of ESBL-Sal.
In summary, we investigated the prevalence and genetic characteristics of ESBL-Sal strains isolated from retail meat in South Korea between 2018 and 2019.The findings suggest that poultry might be the main reservoir for ESBL-Sal, extending beyond Salmonella spp.The predominant ESBL-Sal type identified was Enteritidis-ST11, possessing two plasmid replicons and various antibiotic and virulence factor genes.This finding is significant given previous reports of an outbreak in South Korea involving IncFII plasmids carrying the bla CTX-M-15 gene in Enteritidis-ST11 isolated from humans [21].Thus, retail chicken meat may serve as a potential contagion channel for ESBL-Sal poisoning.Accordingly, continuous monitoring of the poultry industry is essential to prevent the spread of ESBL-Sal.Moreover, further surveillance of ARBs in the supply chain of livestock products sold at retail outlets is warranted.